Vascular extracellular signal-regulated kinase mediates migraine-related sensitization of meningeal nociceptors

Abstract

Objective: To examine changes in the response properties of meningeal nociceptors that might lead to migraine pain and examine endogenous processes that could play a role in mediating them using a clinically relevant model of migraine triggering, namely infusion of the nitric oxide (NO) donor nitroglycerin (NTG).

Methods: Single-unit recordings made in the trigeminal ganglion of rats were used to test changes in the activity and mechanosensitivity of meningeal nociceptors in response to administration of the migraine trigger NTG or another NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) at doses relevant to the human model of migraine headache. Immunohistochemistry and pharmacological manipulations were used to investigate the possible role of meningeal vascular signaling in mediating the responses of meningeal nociceptors to NO.

Results: Infusion of NTG promoted a delayed and robust increase in the mechanosensitivity of meningeal nociceptors, with a time course resembling the development of the delayed migraine headache. A similar sensitization was elicited by dural application of NTG and SNAP. NTG-evoked delayed meningeal nociceptor sensitization was associated with a robust extracellular signal-regulated kinase (ERK) phosphorylation in meningeal arteries. Pharmacological blockade of meningeal ERK phosphorylation inhibited the development of NTG-evoked delayed meningeal nociceptor sensitization.

Interpretation: The development of delayed mechanical sensitization evoked by the migraine trigger NTG is potentially of great importance as the first finding of a neurophysiological correlate of migraine headache in meningeal nociceptors. The arterial ERK phosphorylation and its involvement in mediating the NTG-evoked delayed sensitization points to an important, yet unappreciated, role of the meningeal vasculature in the genesis of migraine pain.

FIGURE 1

NTG infusion evokes a delayed mechanosensitization of a C-unit meningeal nociceptor. (A) Experimental setup (left) and the identification and isolation of a C-unit meningeal nociceptor based on the response latency (16 milliseconds) to electrical stimulation of the dura and a consistent waveform (right). (B) Peri-stimulus time histograms depicting the responses to threshold (S1) and suprathreshold (S2-3) mechanical stimulation of the dura at baseline, before NTG infusion, and at 3 and 6 hrs post infusion. The numbers in parenthesis indicate mean responses to mechanical stimuli in spikes/sec. Note the progressive increase in threshold (S1) responsiveness and the lack of increases in ongoing discharge (between trials) in response to NTG. RF, receptive field; TS, Transverse sinus; TG, Trigeminal ganglion.

FIGURE 2

(A) Time course changes in the average responses of Aδ and C-units meningeal nociceptors in response to NTG infusion. Note that in response to NTG infusion, the Aδ and C-units had a similar delayed increase in both threshold and suprathreshold responses, but did not exhibit an increase in their ongoing discharge rate. (B) Time course changes in the responses of meningeal nociceptors following infusion of the NTG vehicle (6 Aδ, 6 C-units) against a combined re-plotting of the Aδ and C-units responses to NTG from the same 16 neurons plotted in Fig 2A.

FIGURE 3

Time course changes in threshold and suprathreshold responses following topical meningeal application of NTG (5 Aδ, 6 C), SNAP (6 A-δ, 7 C) or their vehicle (4 Aδ, 5 C). Note the development of the delayed sensitization similar to that seen following NTG infusion.

FIGURE 4

NTG infusion promotes ERK activation in dural arteries. Immunohistochemical localization of pERK-immunoreactivity in the dura mater of animals infused with vehicle (A) or NTG (B,C). Note the increased expression of pERK in the walls of the middle meningeal artery (MMA) and the second and third-order arterioles that arise from it (B). Increases in the number of medium size vessels expressing pERK was also found in dural territories remote from the MMA (C). (D) An example of a dural vessel showing pERK-immunoreactivity, which is also labeled with antibody against the arterial marker Ephrin B2 (E). (F) Co-localization (yellow) is shown in (F). The Average±SEM number of pERK labeled dural blood vessels observed per visual field at 1, 2 and 4 hrs following NTG infusion. Note the inhibitory effect induced by local application of the MEK inhibitor U0126 (* p < 0.05, Mann Whitney test NTG vs. vehicle).

FIGURE 5

NTG-evoked delayed sensitization of meningeal nociceptors sensitization involves pERK. Changes in threshold (A) and suprathreshold (B) responses of meningeal nociceptors to mechanical stimulation of the dura 180 and 360 min following infusion with NTG alone (7 Aδ, 9 C), NTG infusion followed by local MEK inhibition using U0126 (6 A-δ, 6 C) or PD98059 (3 A-δ, 5 C) or local treatment with U0126 (4 A-δ 5C) or PD98059 (4 A-δ 5C) alone. (*p < 0.05, Mann Whitney test NTG alone vs NTG+MEK inhibitors).